Whole blood collected from volunteer donors for transfusion recipients is typically separated into its components, red blood cells, platelets, and plasma, by apheresis or other known methods. Each of these fractions are individually stored and used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia, the concentrated platelet component is used to control bleeding, and the plasma component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.
In the United States, blood storage procedures are subject to regulation by the government. The maximum storage periods for the blood components collected in these systems are specifically prescribed. For example, whole blood components collected in an “open” (i.e., non-sterile) system must, under governmental rules, be transfused within twenty-four hours and in most cases within six to eight hours. By contrast, when whole blood components are collected in a “closed” (i.e., sterile) system the red blood cells can be stored up to forty-two days (depending upon the type of anticoagulant and storage medium used) and plasma may be frozen and stored for even longer periods. Platelets can be frozen with dimethyl sulfoxide (DMSO) and stored for years (Valeri et al. (2000) Chapter 6, Frozen Platelets, pages 105–130, in Platelet Therapy: Current Status and Future Trends, Eds. Seghatchian, J. et al., Elsevier, Amsterdam). With 6% DMSO platelets can be stored for about three years at −80° C. and with 5% DMSO for about two years at −150° C.
While red cells are stored in the cold, Murphy and Gardner, New Eng. J. Med. 280:1094 (1969), demonstrated that platelets stored as platelet-rich plasma (PRP) at 22° C. possessed a better in vivo half-life than those stored at 4° C. Thus, more acceptable platelet concentrates could be transfused after storage at room temperature. Until recently, the rules allowed for platelet concentrate storage at room temperature for up to seven days (depending upon the type of storage container). However, it was recognized that the incidence of bacterial growth and subsequent transfusion reactions in the recipient increased to unacceptable levels with a seven-day-old platelet concentrate. Platelet concentrates may currently be stored for no more than five days.
Contamination of blood supplies with infectious microorganisms such as malaria, West Nile virus, HIV, hepatitis and other viruses and bacteria presents a serious health hazard for those who must receive transfusions of whole blood or administration of various blood components such as platelets, red cells, blood plasma, Factor VIII, plasminogen, fibronectin, anti-thrombin III, cryoprecipitate, human plasma protein fraction, albumin, immune serum globulin, prothrombin complex, plasma growth hormones, and other components isolated from blood. Blood screening procedures may miss contaminants, and sterilization procedures, which do not damage cellular blood components but effectively inactivate all or reduce infectious viruses and other microorganisms, are needed in the art. Systems that use the same chemistry to inactivate or reduce microorganisms in different fluids, for example separate blood components, are desired for many reasons, including ease of use in a blood bank setting. It is also desired that the inactivation or reduction treatment be easily implemented in a blood bank setting, and produce inactivation or reduction in a short period of time.
Bacteria can easily be introduced to blood components by at least two different means. First, if the donor is experiencing a mild bacteremia, a condition comprising bacteria in the blood, the blood will be contaminated, regardless of the collection or storage method. Adequate donor histories and physicals will decrease but not eliminate this problem. See B. J. Grossman et al., Transfusion 31:500 (1991).
A second, more pervasive source of contamination is the venepuncture employed when drawing blood. Even when “sterile” methods of skin preparation are employed, it is extremely difficult to sterilize the crypts around the sweat glands and hair follicles. During venepuncture, this contaminated skin is often cut out in a small “core” by a sharp needle. This core can serve to “seed” the blood bag with bacteria that may grow and become a risk to the recipient.
Indeed, many patients requiring platelet transfusions lack host-defense mechanisms for normal clearing and destruction of bacteria because of either chemotherapy or basic hematologic disease. The growth of even seemingly innocuous organisms in stored platelets can, upon transfusion, result in recipient reaction and death. See e.g., B. A. Myhre, JAMA 244:1333 (1980) and J. M. Heal et al., Transfusion 27:2 (1987).
It has been found that platelets which have been treated with a photosensitizer and light to inactivate or reduce pathogens which may be present may show re-activation of pathogens during long-term storage after such a treatment. In addition to platelet aggregation, platelets may show high activation and low extended shape change response by day 5 of storage, both of which may be indications of cytoskeletal changes in the platelets. Such changes may be indications of platelet damage due to the storage conditions. It is therefore necessary to improve the quality of stored photoradiated platelets.
There is a need for methods allowing for better pathogen reduction and/or inactivation while maintaining cell quality above acceptable limits and for methods allowing for improved cell quality while maintaining pathogen reduction and/or inactivation. Large quantities of blood and blood products are discarded by blood banks after certain periods of storage due to expiration of the blood and blood products. By improving the cell quality of blood components during storage and after pathogen reduction and/or inactivation, the shelf life blood components is increased.
In cells, food is oxidized to produce high-energy electrons that are converted to stored energy. This energy is stored in high-energy phosphate bonds in ATP. Ingested sugars are broken down by enzymes that split them into a six-carbon molecule called glucose. Glucose may also be provided to cells in media or storage solutions. The breakdown of glucose to provide energy to cells is an important mechanism in cellular metabolism. This mechanism, known as glycolysis, produces ATP (adenosine triphosphate) in the presence or absence of oxygen. The production of ATP is essential for cellular energy metabolism. Glucose enters the cell by special molecules in the membrane called “glucose transporters.” Once inside the cell, glucose is broken down to make ATP in two pathways. The first pathway requires no oxygen and is called anaerobic metabolism. Anaerobic metabolism or glycolysis occurs in the cytoplasm outside the mitochondria. During glycolysis, glucose is broken down into pyruvate, a three-carbon molecule. This conversion involves a sequence of nine enzymatic steps that create phosphate-containing intermediates. Each reaction is designed to produce hydrogen ions (electrons) that can be used to make energy in the form of ATP. Only two ATP molecules can be made by one molecule of glucose run through this pathway. This pathway is also used to produce two lactate molecules from every one glucose molecule.
For most animal cells, glycolysis is merely the first stage in the breakdown of sugar into cellular energy, since the pyruvic acid that is formed at the last step quickly enters the cell's mitochondria to be completely oxidized to CO2 and H2O in the citric acid cycle. The citric acid cycle is also known in the art as the Kreb's cycle or the tricarboxylic acid (TCA) cycle. The citric acid cycle occurs in the mitochondria and is the common pathway to completely oxidize fuel molecules, which are mostly acetyl CoA, the product from the oxidative decarboxylation of pyruvate. Acetyl CoA enters the cycle and passes through ten steps of reactions that yield energy (ATP) and CO2.
In the case of organisms which are anaerobic (those that do not use molecular oxygen) and for tissues like skeletal muscle that can function under anaerobic conditions, glycolysis is a major source of the cell's ATP. This also occurs in an aerobic cell if the mitochondria of the cell are damaged in some way, thereby preventing the cell from entering the citric acid cycle.
Since ATP is essential to continued cell function, when aerobic metabolism is slowed or prevented by lack of oxygen, anaerobic pathways for producing ATP are stimulated and become critical for maintaining cell viability. Here, instead of being degraded in the mitochondria, the pyruvate molecules stay in the cytosol and can be converted into ethanol and CO2 (as in yeast) or into lactate (as in muscle).
Lactate accumulation in cells causes an increased concentration of hydrogen ions and a decrease in pH. Blood cells in storage that experience a decrease in pH may be only undergoing glycolysis. Such a drop in pH indicates as well as contributes to a decrease in cell quality during cell storage.
Factors which cause cells to enter glycolysis and thereby accumulate lactic acid or lactate include events which occur internally in a body such as strokes or infarctions, as well as external events such as treatment of the cells after removal from a body. One example of an external treatment which might cause cells to accumulate lactate is a procedure to inactivate or reduce pathogens which might be contained in cells or fluids containing cells to be transfused into a recipient. Currently used methods to sterilize pathogenic contaminants which may be present in blood or blood components can cause damage to the mitochondria of the cells being treated. If this occurs, the cells can only make ATP through the glycolysis pathway, causing a buildup of lactic acid in the cell and a subsequent drop in pH during storage.
Mitochondria are critical subcellular organelles of blood components. They are involved in aerobic energy metabolism and the oxidative reactions therein. Mitochondria are sensitive to endogenous and exogenous influences and may be easily damaged or destroyed. Dysfunctional energy metabolism and, more severely, damaged mitochondria, lead to a decline in platelet quality and eventual cell death.
Possible causes of damage to blood components may be storage, pathogen inactivation, and pathogen reduction processes. A reason for changes in platelet viability after pathogen reduction or inactivation may be that irradiation of the platelets to kill pathogens may be causing damage to the platelet mitochondria (Chavez et al. (September 1998) Biochem. Mol. Biol. Int. 46(1):207–214; Masaki et al. (March 1997) J. Dermatol. Sci. 14(3):207–216; and Salet et al. (April 1995) Int. J. Radiat. Biol. 67(4):477–80). It has been observed that a side effect of a pathogen reduction process is that when platelets are subjected to UV light, the mitochondria of the platelets have a greater chance of suffering at least some damage than when they have been subjected to visible light. Mitochondria are present in all oxygen-utilizing organisms in which energy in the form of adenosine triphosphate (ATP) is generated and oxygen is reduced to water. Ninety percent of the oxygen taken in by the organism is consumed by the mitochondria. A substantial byproduct of ATP generation is the formation of potentially toxic oxygen radicals. For example, it is estimated that 1–2% of all reduced oxygen yields superoxide (O2.) and hydrogen peroxide (H2O2). Other reactive oxygen species (ROS) that form are singlet oxygen (O2) and hydroxyl radicals (.OH). Under stress conditions in the cell this can rise to 10% of all consumed oxygen. Mitochondrial membranes are sensitive to lipid peroxidation and depolarization resulting from these ROS.
Furthermore, photochemical methods for pathogen inactivation and reduction of blood products which generate singlet oxygen species in the process of photolysis of the photosensitizer cause further damage to mitochondrial membranes. It is therefore necessary to protect platelet mitochondria of platelets and other blood cells from ROS generated by both photochemical decontamination and stress conditions of storage.
There is a need in the art for methods to prevent damage to mitochondria, to reduce damage to and degradation of blood components during storage and before, during, and after pathogen inactivation and reduction procedures.
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